Supporting data

Argonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in <em>Drosophila melanogaster</em> (DmAgo2) harbors a long and unique N-terminal region. Previous <em>in vitro</em> biochemical studies have shown that the loss of this region does not impair the RNA silencing activity of the complex. However, an N-terminal mutant of <em>Drosophila melanogaster</em> has demonstrated abnormal RNA silencing activity. To explore the causes of this discrepancy between <em>in vitro</em> and <em>in vivo</em> studies, we investigated the biophysical properties of the region. Because the N-terminal region is highly rich in glutamine and glycine residues, which is a well-known property for prion-like domains (PrLD), the possibility of the N-terminal region functioning as a PrLD was tested. Our biochemical assays demonstrated that the N-terminal region can form aggregates that are not dissociated even in the presence of SDS. Also, the aggregates enhanced the fluorescence intensity of thioflavin-T, an amyloid detection reagent. The kinetics of the aggregation followed that of typical amyloid formation exhibiting the self-propagating activity. Further, we directly visualized the aggregation process of the N-terminal region under fluorescence microscopy and found that the aggregations took fractal or fibril shapes. Together, the results indicate that the N-terminal region is a PrLD. Many other PrLDs have been reported to modulate the function of proteins through their aggregation. Therefore, our results raise the possibility that aggregation of the N-terminal region regulates the RNA silencing activity of DmAgo2.